Date post: | 21-Feb-2017 |
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Lab in APS - Confusion over consensus
Dr Vandana KamathChristian Medical College
Vellore
LA testing
Tests Case 1 Case 2 Case 3 History 34 year old
-H/o recurrent abortions
50 year old gentleman during a routine preoperativework up
24 year young man with history of DVT – now on Warfarin
APTT(reference range: 24-32secs)
60 secs 30 secs 67secs
Actin FS (reference range: 22-32secs)
30 secs - 43 secs
Ratio 2 1.5PT(reference range:9-13.5secs /INR
- 49.4/2.8 32secs/2.8
Results LA detected
? Factor deficiency F II, VII,IX,X – in normal range
LA detected
Tests Case 1
History h/o recurrent abortions
APTT 60 secs
Actin FS 30 secs
Recommendations of Scientific and Standardisation Committee of the ISTH (1995,2009,2012)
1. Prolongation of phospholipid dependant clotting test time .
3. Correction in the presence of excess phospholipids
Tests Case 1 History h/o recurrent abortions
APTT( highly sensitive) 60 secs
Mix (1/2 pt+1/2 control) 50 secs
Actin FS(less sensitive) 30 secs
Result Markedly positive LA
Recommendations of Scientific and Standardisation Committee of the ISTH (1995,2009,2012)
2. Failure to correct the prolonged clot time on mixing with normal control plasma and repeating the test.
4. Exclusion of coagulopathies.
Case- 2 Routine pre-operative work up
APTT 30secs
PT/ INR(ISI- 1 )
49.4/2.8
? Factor deficiency
F II, VII,IX,X levels with normal range.
Case- 2 Routine pre-operative work up
APTT (reference range: 24- 30secs)
30secs
PT/ INR(ISI- 1 )Reference range : (9-13.5secs)
49.4/2.8
F II, VII,IX,X levels with normal range.
PT -1 mix with control plasma 46.2secs
? inhibitor
Case- 2 Routine pre-operative work up
APTT 30secs
PT-1/ INR(ISI- 1 )
49.4/2.8
PT -1 mix with control plasma 46.2secs
F II, VII,IX,X levels with normal range.PT -2 (Rabbit brain thromboplastin)
13.4secs
Result LA specific recombinant thromboplastin
Gamma carboxylation of Gla by Vit K --- increasing Negative Charge
VII
IX
X
II
Increased affinity for calcium providing nett strong positive charge..
VII
Ca Ca Ca+IX
Ca Ca Ca+ X
Ca Ca Ca+ II
Ca Ca Ca+
VII
Ca Ca Ca+PE PI PS
IX
Ca Ca Ca+PE PI PS
X
Ca Ca Ca+PE PI PS
II
Ca Ca Ca+PE PI PS
..to bind anionic
phospholipids.
..on the surface of activated platelets
Sensitivity of PT reagentsISI: International sensitivity index.
ISI = 1/ sensitivity
INR: International normalised ratio.
MNPT: Mean normal pooled plasma.
INR = (PT of patient/ MNPT)ISI.
PT- 1 (ISI- 1) more sensitive.
PT-2 (Rabbit brain thromboplastin) (ISI-1.8)- less sensitive.
LA testing
Tests Case 3 History Patient on Warfarin
dRVVT (screen) 67 secs
dRVVT(confirm) 43 secs
Screen/confirm 1.56
PT-1 /INR(ISI -1)
32 secs/ 2.8
PT-2/INR(ISI-1.8)
20.7secs/2.8
PT-1/PT-2 1.56
Result LA detected
LA testing Tests Case 3
History Patient on Warfarin
dRVVT (screen) 67secs
Mixing study 32 secs
dRVVT (confirm) 43secs
Screen/confirm 1.5
PT-1 /INR(ISI -1)
32 secs/ 2.8
PT-2/INR(ISI-1.8)
20.7secs/2.8
Result (referral lab) LA not detected
Recommendations of Scientific and Standardisation Committee of the ISTH (1995,2009,2012)
1. Prolongation of phospholipid dependant clotting test time .
2. Failure to correct the prolonged clot time on mixing with normal control plasma and repeating the test.
3. Correction in the presence of excess phospholipids
4. Exclusion of coagulopathies.
Mixing studies
Reporting a prolonged or a positive screening tests (either APTT or dRVVT) as lupus anticoagulant without mixing studies which is the only way to demonstrate the inhibition or the anticoagulant effect of Lupus anticoagulant is unethical and against the standard as it reports a lot of false positives consistently.
Indications of LA testingPart of all thrombotic work ups.An unexpectedly prolonged screening APTT
may also trigger an LA investigation.
Recommendations of Scientific and Standardisation Committee of the ISTH (1995,2009,2012)
1. Prolongation of phospholipid dependant clotting test time .
2. Failure to correct the prolonged clot time on mixing with normal control plasma and repeating the test.
3. Correction in the presence of excess phospholipids
4. Exclusion of coagulopathies.
LA - SSC-ISTH consensus criteriaNo single PL dependent clotting test is 100%
sensitive to detect LARecommended to perform at least one
additional screening clotting test Category 1 Screening test - activation of
Intrinsic pathway – aPTT or KCTCategory 2 Screening test - direct activation
of clotting – dRVVT or dPT (TTI)Confirmatory tests – dRVVT kit comes with
extra phospholipid. Few aPTT reagents – hexagonal PL.
Simple Confirmatory for aPTT, dPT, and KCT make PRP and Freeze thaw to make platelet phospholipid available – then do these tests.
QC – reference range to be established
Laboratory tests should employ A detection or screening stage
(prolongation of the clotting time) – SCREENING TESTS – reduced levels of PL.
• Failure of correction of the prolongation when normal plasma is added in order to exclude factor deficiency as the cause of the prolongation – MIXING STUDIES.
• A confirmation stage showing that the prolongation is phospholipid dependent, for example by showing that addition of excess phospholipid corrects the clotting time – CONFIRMATORY TESTS
Mixing/Correction Studies
Done when PT/APTT are prolongedMixing studies :- Mix equal volumes of test
(abnormal) plasma and control pool plasma (where all factors are present in normal quantity) and repeat the test.
Rationale: - if the prolongation in time is due to deficiency of
factor (s) in the test plasma, normal plasma will provide the deficient factor when they are mixed correcting the prolonged time
- if the prolongation in time is due to an inhibitor (antibody to factor or heparin) the normal plasma will also be inhibited and the prolonged time will remain prolonged
Correction of time: Deficiency of factorNo correction: Inhibitor
No correctionWhatever caused the abnormal
(prolonged) timing is also causing an abnormality to normal plasma after the patients plasma was mixed in it.
- Heparin - Lupus anticoagulant
(antiphospholipid antibody) - Factor Inhibitor ( antibodies to
Clotting factors) - FDP/D-Dimer
Correction StudiesDirect interpretation to therapyPatient who is bleeding with a
prolongation in plasma clotting testsCorrection of the time by mixing studies
in which the normal plasma supplied the deficient factor will directly mean that if you give (transfuse) the patient - normal plasma (FFP) his clotting defect, that lead to the prolongation of the time, will get corrected and the bleeding will stop.
Non Correction of time – No benefit of transfusing FFP. Use safer productsHeparin – use ProtaminInhibitor – other agents
Standard Mixing studies – will miss a FVIII inhibitor
APTT – One Month Statistics
aPTT by SynthASiL (N=7174)
27.5% Prolonged aPTT (n=1964)
2nd line aPTT: Actin FS
80% Normal aPTT (n=1460)
72.5% Normal aPTT (n=5183)
Only 7 % remains to be sorted (n=504)
29
APTT STA – PTT A
Normal (within reference range)
Release report withReference range.
APTT Abnormal (more than upper limit of ref. range)
Normal(within ref. range)
Release APTT FS with its ref. range
Abnormal-do APTT mix with STA – PTT A
Release APTT STA-PTT A result with Mix
Do APTT FS